Oligonucleotides for genotyping thymidylate synthase gene

ABSTRACT

Oligonucleotides for genotyping the thymidylate synthase gene are provided. The number of tandem repeats in the promoter region of the thymidylate synthase gene can be identified based on the hybridization of an oligonucleotide of the invention to the genomic DNA of a subject. Therefore, the genotype of the thymidylate synthase gene can be identified based on the number of tandem repeats. The genotype relates to the responsiveness of a subject towards an antitumor agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/529,713, filed on Mar. 29, 2005, now U.S. Pat. No. 7,919,235, whichis the National Stage of International Application No.PCT/JP2002/010167, filed Sep. 30, 2002. The contents of theseapplications are incorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to the genotyping of the thymidylatesynthase gene. The present invention also relates to the prediction ofthe responsiveness of a subject towards an antitumor agent based on thethymidylate synthase genotype.

BACKGROUND

5-Fluorouracil (5-FU) is a compound that has been utilized as anantitumor agent for a long time (Heidelberger C, Chaudhuri N K,Danenberg P V, Mooren D, et al. (1957) Fluorinated pyrimidines: a newclass of tumor inhibitory compounds. Nature 179:663-666). The antitumoreffects of 5-FU against various tumors have been reported.

The cytotoxic effect of 5-FU is based on the inhibition of DNA synthesisin cells. 5-FU inhibit even the DNA synthesis in non-tumor tissue notonly that in tumor tissue. However, since usually a far more active DNAsynthesis takes place in tumor tissues compared to non-tumor tissues,the manifested influence of the 5-FU-mediated inhibitory action isthought to be comparatively larger in tumor tissues. It is through thismechanism that 5-FU exerts an inhibitory action on tumor tissues.

On the other hand, the administration of 5-FU, which is a cytotoxicagent, often accompanies adverse effects that cannot be ignored. Thecytotoxic effect of 5-FU disables not only tumor tissues, but alsonon-tumor tissues. 5-FU sensitivity in 5-FU administered patients isconsidered to be closely related to the magnitude of the adverse effectsof the drug.

5-FU is a DNA synthesis inhibitor that targets thymidylate synthase.Thymidylate synthase catalyzes the intracellular conversion ofdeoxyuridylate to deoxythymidylate. Deoxythymidylate is the only de novosource of thymidylate, an essential precursor for DNA synthesis(Danenberg PV (1997) Thymidylate synthase. a target enzyme in cancerchemotherapy. Biochim Biophys Acta 473:73-92).

The promoter of thymidylate synthase gene has been demonstrated to bepolymorphic (Nobuyuki H, Masahiko C, Ryushi N, Keiichi T (1993)Characterization of the regulatory sequences and nuclear factors thatfunction in cooperation with the promoter of the human thymidylatesynthase gene. Biochim Biophys Acta 1216:409-416). Furthermore, it hasbeen shown that the polymorphism of the thymidylate synthase genepromoter is related to the response of a subject towards 5-FU. Humanthymidylate synthase gene has a polymorphism comprising two or threetandem repeats of a 28-bp sequence in its regulatory region. Theexpression level of the thymidylate synthase gene which is homozygousfor three tandem repeats, is 3.6 times that of the thymidylate synthasegene which is homozygous for two tandem repeats. As a result, subjectscarrying the three tandem repeats have significantly fewer adverseeffects (Pullarkat, S T, Stoehlmacher J, Ghaderi V, Xiong Y, et al.(2001) Thymidylate synthase gene polymorphism determines clinicaloutcome of patients with colorectal cancer treated with fluoropyrimidinechemotherapy. Pharmacogenomics J 1:65-70).

Thymidylate synthase is an important target of not only 5-FU, but alsoother antitumor agents. For example, capecitabine, which was developedas an oral prodrug of 5-FU, also targets thymidylate synthase. Thissuggested that the polymorphism in the regulatory region of the humanthymidylate synthase gene is a useful marker for determining theresponsiveness of a subject towards antitumor agents.

SUMMARY

An objective of the present invention is to provide a method forgenotyping the thymidylate synthase gene. Especially, oligonucleotidessuitable for genotyping the thymidylate synthase gene are provided.Another objective of the present invention is to provide a method forpredicting the responsiveness of a subject towards an antitumor agentthat targets thymidylate synthase based on the thymidylate synthasegenotype.

It has been demonstrated that the polymorphism of tandem repeats in thepromoter region of the thymidylate synthase gene is related to theresponsiveness of a subject against antitumor agents that targetthymidylate synthase. Therefore, the effectiveness or the degree ofadverse effects of an antitumor agent can be predicted by analyzing thispolymorphism. Polymorphism is generally determined by amplifying genomicDNA and analyzing amplicon size. The size of amplicons amplified by PCRis analyzed by gel electrophoresis. However, gel electrophoresis is alaborious and time-consuming analytical technique. Antitumor agents thattarget thymidylate synthase are important drugs in the chemotherapy ofcancer. Therefore, a method that more conveniently yields informationregarding the responsiveness of a subject against an antitumor agentthat targets thymidylate synthase is desired.

Extensive research was carried out by the present inventors on a methodfor identifying the number of tandem repeats in the promoter region ofthe thymidylate synthase gene. As a result, they discovered that thenumber of tandem repeats in genomic DNA can be identified by using anoligonucleotide having a specific nucleotide sequence as a probe, anddetecting mismatches therein. Furthermore, the present inventorsconfirmed that the genotype of the subject could be determined based onthe number of tandem repeats elucidated as above. Furthermore, thepresent inventors discovered that it is possible to design a strategyfor treating a cancer in a patient by relating thymidylate synthasegenotype, which is determined by the present invention, with theresponsiveness of the subject against antitumor agents targetingthymidylate synthase.

Namely, the present invention provides an isolated oligonucleotide that

-   -   (a) comprises a nucleotide sequence that is complementary to a        region consisting of:        -   (i) the central repeat unit of three repeat units composing            a tandem repeat in the promoter region of the thymidylate            synthase gene, and        -   (ii) the repeat unit located downstream of the central            repeat unit, and    -   (b) hybridizes to the region of (a) under highly stringent        hybridization conditions.

As mentioned earlier, the tandem repeat in the promoter region of thethymidylate synthase gene is polymorphic. Namely, the presence of twokinds of tandem repeats, a tandem repeat consisting of two repeat units,and a tandem repeat consisting of three repeat units, has beenelucidated. “Tandem repeat” as mentioned herein refers to a region inwhich two or more similar nucleotide sequences repeat successively.Similar repeating nucleotide sequences are called repeat units.Generally, the number of repeats is 2 or more. In the present invention,the number of repeats to be identified is 2 and 3. Hereinafter, apolymorphic form in which three repeat units compose a tandem repeatwill be referred to as 3R. Furthermore, a polymorphic form in which tworepeat units compose a tandem repeat will be referred to as 2R. Thesepolymorphic form nucleotide sequences can be found in a DNA database(3R: GenBank accession number AF279906, 2R: GenBank accession numberAF279907). An oligonucleotide of this invention has a nucleotidesequence that is complementary to the nucleotide sequence constituting aregion comprising two units of these polymorphic forms, which are thecentral repeat unit of 3R, and the repeat unit located downstream of thecentral repeat unit. More specifically, 132-193 of the nucleotidesequence disclosed in GenBank Accession No. AF279906 is the regionindicated in the above-mentioned (a). In the present invention, thecomplementary nucleotide sequences specifically include the followingtwo nucleotide sequences:

(1) a nucleotide sequence determined to be complementary to a certainnucleotide sequence according to the Watson-Crick rule, or

(2) a nucleotide sequence having a homology of 80% or more with thenucleotide sequence of (1).

Preferably, (2) includes a nucleotide sequence having a homology of 90%or more, more preferably, 95% or more, and even more preferably, 97% ormore with the nucleotide sequence of (1). Algorithms for determiningnucleotide sequence homology are well known. For example, programs forcalculating nucleotide sequence homology using BLAST are in practicaluse. These programs can be used via the Internet.

The present inventors completed this invention by discovering that thetwo polymorphic forms can be distinguished when an oligonucleotidehaving such a nucleotide sequence is hybridized to genomic DNA under thesame conditions. That is, the oligonucleotide hybridizes to a 3R tandemrepeat, but does not hybridize to 2R under the same conditions.

Furthermore, the oligonucleotide of this invention hybridizes to theregion under highly stringent hybridization conditions. In the presentinvention, “highly stringent hybridization conditions” can be achievedby simultaneously fulfilling the following conditions of (1) and (2).Incidentally, “does not substantially hybridize” means that nohybridization is detected under the same conditions as (1) describedbelow:

(1) a certain oligonucleotide hybridizes to the region of (a), and

(2) the oligonucleotide does not substantially hybridize to the tandemrepeat consisting of two repeat units, which is another polymorphic formof the gene.

In the present invention, preferable oligonucleotide hybridizes to the3′ end repeat unit of the two repeat units composing a tandem repeat inthe promoter region of the thymidylate synthase gene, underhybridization conditions that are less stringent than (b). Theoligonucleotide is useful for the melting curve analysis of the presentinvention.

The oligonucleotide fulfilling the above-mentioned conditions issometimes referred to as a mutation probe in this invention. The repeatunits constituting the promoter of the thymidylate synthase gene are notcompletely identical. The nucleotide sequence of each of the threerepeat units composing a tandem repeat in the promoter region of thethymidylate synthase gene is shown below.

-   5′-ccgcgccacttggcctgcctccgtcccg ccgcgccacttcgcctgcctccgtcccg    ccgcgccacttcgcctgcctccgtcccccgcccg-3′ (SEQ ID NO:5)    Therefore, an oligonucleotide that hybridizes to a specific repeat    unit may not hybridize to other repeat units. The oligonucleotide of    this invention was designed by utilizing such a phenomena. A    preferable oligonucleotide of this invention is an oligonucleotide    comprising the nucleotide sequence of SEQ ID NO: 1. A method for    synthesizing an oligonucleotide having a nucleotide sequence of    interest is known to those skilled in the art.

The oligonucleotides of this invention can be used to identify thenumber of tandem repeats in the promoter region of the thymidylatesynthase gene. That is, the present invention relates to a method foridentifying the number of tandem repeats in the promoter region of thethymidylate synthase gene comprising the steps of:

-   -   (a) amplifying a genomic DNA that comprises tandem repeats in at        least the promoter region of the thymidylate synthase gene,    -   (b) hybridizing the oligonucleotide of the present invention to        the amplified genomic DNA of step (a) under stringent        conditions,    -   (c) detecting a hybridization between the oligonucleotide and        the genomic DNA, and    -   (d) identifying the number of tandem repeats as “two” when a        hybridization is not detected, identifying the number of tandem        repeats as “three” when a hybridization is detected.

Preferably, the method of present invention further comprising:

-   -   (e) hybridization the oligonucleotide of the present invention        to the amplified genomic DNA of step (a) under hybridization        conditions that are less stringent than (b),    -   (f) detecting a hybridization between the oligonucleotide and        the genomic DNA, and    -   (g) identifying the number of tandem repeats as “two” when        hybridization is not detected in (c) but is detected in (f)

In the present invention, genomic DNA can be obtained from a biologicalsample from a subject whose number of tandem repeats in the promoterregion of the thymidylate synthase gene is to be identified. Forexample, a method for obtaining genomic DNA from blood cells collectedfrom a subject is well known. Any method that can amplify DNA in anucleotide sequence specific manner can be utilized to amplify genomicDNA. Generally, the PCR method is used to amplify genomic DNA. Whenamplifying DNA, it is sufficient to amplify an arbitrary regioncontaining tandem repeats in at least the promoter region of thethymidylate synthase gene. More specifically, genomic DNA of at least 90by that contains tandem repeats can be selected as the region to beamplified. For example, when detecting hybridization by melting curveanalysis using LightCycler as described below, the length of the DNA tobe amplified is usually 700 by or less.

The method of this invention for identifying the number of tandemrepeats in the promoter region of the thymidylate synthase gene includesthe step of hybridizing the mutation probe to the amplified genomic DNAunder stringent conditions. Among the polymorphic forms in the promoterregion of the thymidylate synthase gene, the mutation probe hybridizesto 3R, but not to 2R. Therefore, using hybridization of the mutationprobe as an index, the number of tandem repeats can be determined. Todetect the hybridization of the mutation probe, an arbitrary method fordetecting DNA hybridization can be used.

In the present invention, melting curve analysis is the preferred methodfor detecting differences in nucleotide sequences using DNAhybridization. Certain oligonucleotides hybridize to polynucleotideshaving complementary sequences. Although DNA hybridization issequence-specific, it is difficult to completely exclude hybridizationstowards very similar nucleotide sequences. Melting curve analysis is amethod for detecting changes in hybridization based on changes inmelting temperature (Tm). Double strand DNA (dsDNA) formed byhybridization of nucleotide sequences that are complementary to eachother, gradually dissociate and become single strand DNA (ss DNA) whenthe temperature is raised. When the relationship between the change fromds DNA to ss DNA and the change in temperature is plotted on a graph,the change into ss DNA is not linear, and occurs abruptly at a certaintemperature. The temperature at which this abrupt change to ss DNAoccurs is Tm. Tm changes with various factors such as nucleotidesequence, and composition of the solution in which the DNA exists.However, under specific conditions, Tm clearly changes depending on thenucleotide sequence, when there is a difference in a nucleotidesequence. Therefore, differences in Tm of a certain oligonucleotidetowards a target sequence can be detected easily, even if the differencein the target sequence is slight. Melting curve analysis is a methodthat facilitates sensitive detection of slight differences in nucleotidesequences based on differences in Tm detected as above.

To carry out the method of this invention based on melting curveanalysis, the difference in Tms of the mutation probe towards 3R and 2Rcan be detected. In melting curve analysis, the hybridization of amutation probe towards a target sequence must be observed. There are nolimitations on the method for observing hybridization. In the presentinvention, a preferred method for observing hybridization includes theapplication of fluorescence resonance energy transfer (FRET). FRET is amethod for detecting hybridization utilizing the fact that twooligonucleotides that hybridize to adjacent regions on a target sequencecome in close proximity to each other due to hybridization. The ends ofthe two adjacent oligonucleotides are labeled with differentfluorophores that function as a donor or an acceptor. When the two comeinto close proximity due to hybridization, a characteristic fluorescenceemission can be detected due to the energy transfer between thefluorophores.

To apply the FRET to the method of this invention, a secondoligonucleotide that hybridizes to the region adjacent to the mutationprobe is necessary for detecting the hybridization of the mutationprobe. The present inventors discovered that when using as the mutationprobe an oligonucleotide having the aforementioned properties (a) and(b), an oligonucleotide that can hybridize to the 5′ side thereof isuseful as the second oligonucleotide. More specifically, the presentinvention relates to an isolated oligonucleotide that hybridizes to theregion adjacent to the 5′ side of the oligonucleotide that:

-   -   (a) comprises a nucleotide sequence that is complementary to a        region consisting of:        -   (i) the central repeat unit of three repeat units composing            a tandem repeat in the promoter region of the thymidylate            synthase gene, and        -   (ii) the repeat unit located downstream of the central            repeat unit, and    -   (b) hybridizes to the region of (a).

In the present invention, “the region adjacent to the 5′ side of theoligonucleotide” refers to the 5′ side region of the region to which theoligonucleotide hybridizes on the target nucleotide. “Adjacent to”includes the case where the ends of the oligonucleotide and the secondoligonucleotide are −0 to 10 bases apart, and preferably 0 to 5 bases.In the present invention, when using the second oligonucleotide forFRET, it is sometimes called an anchor probe. It is preferred that theTm of the anchor probe is the same or more than the Tm of the mutationprobe towards a 3R tandem repeat. The relationship between genomic DNAand each probe is indicated in FIG. 1.

Hybridization of the mutation probe can be observed by FRET whileperforming PCR. That is, hybridization of the mutation probe can bedetected while amplifying genomic DNA. To detect hybridization of themutation probe during PCR, it is preferable to design the Tm of themutation probe and anchor probe in such a way that hybridization to theamplicon takes place during the annealing phase of PCR. To adjust the Tmto an appropriate range, mismatched bases can be included in thenucleotide sequences of the mutation probe and the anchor probe.Furthermore, it is preferred that the 3′ end of each probe is modifiedto avoid extension of the probes by DNA polymerase. For example, anoligonucleotide labeled at its 5′ end with a fluorophore can be modifiedat its 3′ end by phosphorylation.

An instrument that uses FRET to detect hybridization of the mutationprobe during PCR is commercially available. For example, LightCycler™ isequipped with the mechanism and software necessary for analyzing a PCRamplicon by FRET. The present invention can be carried out using such aninstrument. A specific protocol for carrying out the method of thisinvention by LightCycler™ using the mutation probe and the anchor probeis described below.

Genomic DNA that comprises tandem repeats in at least the promoterregion of the thymidylate synthase gene is amplified with specificprimers from human genomic DNA. The amplicon is detected by fluorescenceusing the mutation probe and the anchor probe as a specific pair ofHybridization Probes. The Hybridization Probes consist of two differentshort oligonucleotides that hybridize to an internal sequence of theamplified fragment during the annealing phase of the PCR cycle. Oneprobe (mutation probe) is labeled at the 5′-end with LightCycler-Red640, and to avoid extension, modified at the 3′-end by phosphorylation.The second probe (anchor probe) is labeled at the 3′-end withfluorescein. Only after hybridization to the template DNA do the twoprobes come in close proximity, resulting in fluorescence resonanceenergy transfer (FRET) between the two fluorophores. During FRET,fluorescein, the donor fluorophore, is excited by the light source ofthe LightCycler Instrument, and part of the excitation energy istransferred to LightCycler-Red 640, the acceptor fluorophore. Theemitted fluorescence of LightCycler-Red 640 is then measured by theLightCycler Instrument.

The oligonucleotides of the present invention are also used to determinethe genotype by performing a melting curve analysis after theamplification cycles are completed and the amplicon is formed.

The fluorescein-labeled oligonucleotide of the present inventionhybridizes to a part of the target sequence that is not mutated andfunctions as an anchor probe.

The other oligonucleotide, labeled with Light Cycler-Red640, spans therepeat unit (mutation probe). The latter probe has a lower meltingtemperature (Tm) than the anchor probe, thus ensuring that thefluorescent signal generated during the melting curve analysis isdetermined only by the mutation probe. The Tm is not only dependent onthe length and G+C content, but also on the degree of homology betweenthe mutation probe and the template DNA. When a 2R type tandem repeat ispresent, the mismatch of the mutation probe with the target destabilizesthe hybrid. With a 3R type tandem repeat, mismatches do not occur, andthe hybrid has a higher Tm. The temperature is slowly increased and whenthe mutation probe melts off and the two fluorescent dyes are no longerin close proximity, the fluorescence will decrease. For mutatedgenotypes, this will occur at temperatures lower than that for thewildtype genotype.

The 5R type tandem repeat of the thymidylate synthase has been reportedrecently (Luo H R, Lu X M, Yao Y G, Horie N, Takeishi K, Jorde L B,Zhang Y P. (2002) Length polymorphism of thymidylate synthase regulatoryregion in Chinese populations and evolution of the novel alleles.Biochem Genet 40(1-2): 41-51). The mutation probe of the presentinvention will hybridize to the 5R type tandem repeat besides the 3Rtype one. However, it does not make significant difference whether theprobe can distinguish between the 3R type and 5R type. The genotypingand prediction for responsiveness in the present invention can becarried out whenever the probe can distinguish the 2R type thatpossesses high responsiveness from other polymorphic types.

As described above, the genotype of the thymidylate synthase gene iselucidated based on the number of tandem repeats determined by thepresent invention. More specifically the present invention provides amethod for genotyping the thymidylate synthase gene of a subject, themethod comprising:

-   -   (a) identifying the number of tandem repeats in the promoter        region of the thymidylate synthase gene by the method of present        invention, and    -   (b) determining that the thymidylate synthase genotype of the        subject is “homozygous 2R/2R” when the number of tandem repeats        is identified as only two, “homozygous 3R/3R” when the number of        tandem repeats is identified as only three, or “heterozygous        2R/3R” when the number of tandem repeats is identified as both        “two” and “three”.

There is a report that used the LightCycler for genotyping of othergenes (Nicolas Von Ahsen et al. Clinical Chemistry 46: 12, 1939-1945(2000), DNA base bulge vs. unmatched end formation in probe-baseddiagnostic insertion/deletion genotyping: Genotyping the UGT1A1 (TA)npolymorphism by real-time fluorescence PCR). However, the LightCyclerhas not been used for genotyping the thymidylate synthase gene prior tothe present invention.

Based on the thymidylate synthase genotype elucidated, theresponsiveness of a subject towards an antitumor agent targetingthymidylate synthase can be predicted. More specifically, the presentinvention provides a method for predicting the responsiveness of asubject towards an antitumor agent targeting thymidylate synthase, themethod comprising:

-   -   (a) determining the thymidylate synthase genotype of the subject        by the method of the invention, and    -   (b) associating the thymidylate synthase genotype with the        responsiveness of the subject towards an antitumor agent        targeting thymidylate synthase.

In the present invention, “predicting the responsiveness of a subjecttowards an antitumor agent targeting thymidylate synthase” refers to theprediction of the degree of cytotoxic activity of an antitumor agenttargeting thymidylate synthase towards a certain patient and/or a tumortissue obtained from a patient. As mentioned earlier, thymidylatesynthase genotype is a major factor determining the expression level ofthymidylate synthase. Furthermore, the expression level of thymidylatesynthase is related to the responsiveness of a subject towards anantitumor agent targeting thymidylate synthase. That is, the expressionlevel of thymidylate synthase is inversely correlated to theresponsiveness. Therefore, the genotype and the responsiveness can becorrelated. Specifically, based on the present invention, a subjectwhose thymidylate synthase genotype has been determined to be homozygous2R/2R is predicted to have high responsiveness. That is, in thissubject, the cytotoxic activity of the antitumor agent targetingthymidylate synthase is predicted to be high. On the other hand, asubject whose genotype has been determined to be 2R/3R heterozygous, or3R/3R homozygous is predicted to have a normal responsiveness. That is,in this subject, it is predicted that the antitumor agent targetingthymidylate synthase will have a normal cytotoxic activity “Normalcytotoxic activity” refers to a condition in which the possibility ofhaving severe adverse drug effects is not high when the drug isadministered according to a usual administration protocol. Alternately,it refers to a condition in which the inhibitory action of a drug ontumor tissues cannot be expected unless the dose is based on a normaladministration protocol.

In the present invention, the antitumor agent targeting thymidylatesynthase includes an antitumor agent having an action to adjust theactivity of thymidylate synthase directly or indirectly. One of themodes of action of a 5-FU-type antitumor agent is inhibiting theactivity of thymidylate synthase by its metabolite, FdUMP. Thymidylatesynthase is the direct target enzyme of 5-FU-type antitumor agents. Onthe other hand, responsiveness towards Methotrexate used for treatingleukemia and such, is also thought to be related to the thymidylatesynthase genotype (The Lancet Vol. 359, 1033-1034, Mar. 23, 2002).Methotrexate is an inhibitor of dihydrofolate reductase. On the otherhand, the reaction catalyzed by thymidylate synthase requires reductionof dihydrofolate. That is, Methotrexate is an antitumor agent thatindirectly inhibits thymidylate synthase. The method of this inventionallows the prediction of the responsiveness towards such antitumoragents that have indirect inhibitory actions on thymidylate synthase.Examples of antitumor agents for which the responsiveness can bepredicted by the method of this invention are 5-FU, Carmofur, Tegafur,UFT, S-1, Doxifluridine, Capecitabine, Fludarabine, Methotrexate,Leucovorin, and Levofolinate.

Based on responsiveness determined in this manner, a chemotherapy methodfor cancer can be designed. More specifically, the present inventionrelates to a method for determining the dose and/or the type of anantitumor agent that targets thymidylate synthase for treating a cancerpatient, the method comprising:

-   -   (a) determining the thymidylate synthase genotype of the patient        by the method of the present invention, and    -   (b) for a “homozygous 2R/2R” patient, deciding to: (i)        administer an antitumor agent dose that is lower than the        normally used dose, or (ii) use an antitumor agent that has a        different target.

For patients predicted to have a high responsiveness towards anantitumor agent that targets thymidylate synthase, lowering the dose ofthe antitumor agent, or selecting an antitumor agent having a differenttarget is recommended. As a result, the danger of exposing a patient toadverse drug effects can be decreased.

Additionally, the present invention provides a kit for identifying thenumber of tandem repeats in the promoter region of the thymidylatesynthase gene, the kit comprising:

-   -   (a) an oligonucleotide comprising the nucleotide sequence of SEQ        ID NO: 1, and    -   (b) an oligonucleotide comprising the nucleotide sequence of SEQ        ID NO: 2.

As mentioned earlier, the oligonucleotides constituting the kit of thisinvention can be labeled with a fluorophore for FRET. Furthermore,additional factors can be combined with the kit of this invention.Examples of additional factors are:

hybridization buffer,

control sample that yields the result of 2R and/or 3R, and

DNA polymerase and substances for PCR.

Any patents, patent applications, and publications cited herein areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relationship between tandem repeats of 2R (SEQ ID NO:6)and 3R (SEQ ID NO:7), and two probes that hybridize to the tandemrepeats. The nucleotide sequences in the figure indicate the anchorprobe (top; SEQ ID NO:2), the tandem repeats of genomic DNA (middle),and the mutation probe (bottom; SEQ ID NO:1). The sequences of therepeat units are in italics. Each repeat unit is separated by a space.All sequences are shown as the sequence of the sense strand for easyverification of the sequences. In reality, either one of the genomic DNAand each probe is an antisense sequence.

EXAMPLES

1) Extraction of DNA

Genomic DNA was purified from 100 n1 of human whole blood. For thepurification, GFX™ Genomic Blood DNA Purification Kit (AmershamPharmacia Biotech) was used.

2) Sequences of PCR Primer FW, PCR Primer REV, Hybridization Probe(Anchor), and Hybridization Probe (Mutation)

PCR Forward Primer Sequence (SEQ ID NO: 3)5′-GTG GCT CCT GCG TTT CCC C-3′ PCR Reverse Primer Sequence(SEQ ID NO: 4) 5′-TCC GAG CCG GCC ACA GGC AT-3′Hybridization probe (Anchor) Sequence (SEQ ID NO: 2)5′-CGC GGA AGG GGT CCT GCC ACC GCG CCA CTT GGCCTG CCT CGG TCC CGC CG-FITC-3′ Hybridization probe (Mutation) Sequence(SEQ ID NO: 1) 5′-LCRed640-CTT GGC CTG CCT CCG TCC CGC CGC GCC-phosphorylation-3′

Primers were synthesized by SAWADY Technology Co., Ltd., and probes weresynthesized by Nihon Gene Research Lab's, Inc.

3) Preparation of PCR Mixture

LightCycler-FastStart DNA Master SYBR Green I Kit (Roche Diagnostics)was used. The PCR mixture was prepared from the following compositions.

PCR Grade Distilled Water (attached to Kit) 5.4 μl 10 μM Forward Primer1 μl (final conc. 0.5 μM) 10 μM Reverse Primer 1 μl (final conc. 0.5 μM)4 pmol/μl Hybridization probe (Anchor) 1 μl 4 pmol/μl Hybridizationprobe (Mutation) 1 μl 25 mM MgCl₂ (attached to Kit) 2.4 μl (final conc.4 mM) DMSO 1.2 μl Hybridization master mix (attached to Kit) 2 μl HumanBlood Genomic DNA solution 5 μl Total volume 20 μl4) PCR Using LightCycler

Experimental Protocol for PCR using the LightCycler

Experimental Protocol

Type None Cycles 1 Program Denature Hold 2° Target Step Step SegmentTemperature Time Slope Temp Size Delay Acquisition Number Target (° C.)(sec) (° C./sec) (° C.) (° C.) (Cycles) Mode 1 95 300 20 0 0 0 NoneType Quantification Cycles 33 Program PCR Hold 2° Target Step StepSegment Temperature Time Slope Temp Size Delay Acquisition Number Target(° C.) (sec) (° C./sec) (° C.) (° C.) (Cycles) Mode 1 95 15 20 0 0 0None 2 58 5 20 0 0 0 Single 3 72 12 20 0 0 0 None Type Melting CurveCycles 1 Program Melting Hold 2° Target Step Step Segment TemperatureTime Slope Temp Size Delay Acquisition Number Target (° C.) (sec) (°C./sec) (° C.) (° C.) (Cycles) Mode 1 95 3 20 0 0 0 None 2 77 30 0.5 0 00 None 3 70 30 0.2 0 0 0 None 4 56 30 0.2 0 0 0 None 5 95 0 0.1 0 0 0Continuous Type None Cycles 1 Program Cooling Hold 2° Target Step StepSegment Temperature Time Slope Temp Size Delay Acquisition Number Target(° C.) (sec) (° C./sec) (° C.) (° C.) (Cycles) Mode 1 40 30 20 0 0 0None5) Melting Curve Analysis Using LightCycler

Analysis was performed by using the melting curves program ofLightCycler. Fluorescence was set to F2/F1. The “Calculation method” of“Step 1: Melting Peaks” was set to “Linear with Background Correction”.To adjust the base line, the cursor at the low temperature side (Green)was set to around 62° C. and cursor at the high temperature side was setto around 83° C. To calculate the melting peak area, “Step 2: PeakAreas” was selected, and the number of peaks were chosen for each sampleto obtain the Tm Value, peak area, and standard deviation.

6) Determination

A sequence whose peak Tm value was only 68-70° C. was determined to be2R/2R homozygous, and a sequence whose peak Tm value was only 76-79° C.was determined to be 3R/3R homozygous. A sequence which had both Tmvalues was determined to be 2R/3R heterozygous.

Oligonucleotides for genotyping the thymidylate synthase gene areprovided. The number of tandem repeats in the promoter region of thethymidylate synthase gene can be identified based on the hybridizationof an oligonucleotide to the genomic DNA. The identification based onhybridization is simple and fast compared to gel electrophoresis. Usingthe oligonucleotides of this invention, the number of tandem repeats canbe identified easily using mismatches as indexes.

Therefore, the genotype of the thymidylate synthase gene can bedetermined based on the number of tandem repeats. The genotype relatesto the responsiveness of a subject towards an antitumor agent targetingthymidylate synthase. Therefore, based on the present invention, it ispossible to predict the responsiveness towards an antitumor agenttargeting thymidylate synthase. Furthermore, based on the responsivenesspredicted according to the present invention, a chemotherapy method forcancer can be designed. More specifically, for patients predicted tohave a high responsiveness towards an antitumor agent targetingthymidylate synthase, lowering the dose of the antitumor agent, orselecting an antitumor agent having a different target is recommended.As a result, the danger of exposing a patient to adverse drug effectscan be reduced.

The invention claimed is:
 1. A method for identifying a number of tandemrepeats in the promoter region of a thymidylate synthase gene, themethod comprising: (a) amplifying a genomic DNA that comprises tandemrepeats in at least the promoter region of the thymidylate synthasegene; (b) contacting, under stringent hybridization conditions, theamplified genomic DNA with either (i) a first oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO:1 and a second oligonucleotidecomprising the nucleotide sequence of SEQ ID NO:2, or (ii) a firstoligonucleotide comprising the nucleotide sequence of the exactcomplement of SEQ ID NO:1 and a second oligonucleotide comprising thenucleotide sequence of the exact complement of SEQ ID NO:2, wherein thehybridization conditions are such that hybridization between the firstoligonucleotide and the amplified genomic DNA can be detected if threetandem repeats are present, but not if only two tandem repeats arepresent; (c) detecting whether hybridization between the firstoligonucleotide and the amplified genomic DNA has occurred; and (d)identifying the number of tandem repeats as “two” when hybridization isnot detected.
 2. The method of claim 1, wherein step (c) comprisesmelting curve analysis.
 3. The method of claim 1, wherein the nucleotidesequence of the first oligonucleotide comprises SEQ ID NO:1.
 4. Themethod of claim 1, the method comprising detecting fluorescenceresonance energy transfer using a first fluorescent dye at the 5′ end ofthe first oligonucleotide and a second fluorescent dye at the 3′ end ofthe second oligonucleotide, wherein the first fluorescent dye transfersfluorescence resonance energy to the second fluorescent dye or viceversa.
 5. The method of claim 1, wherein the nucleotide sequence of thefirst oligonucleotide consists of SEQ ID NO:1.
 6. The method of claim 4,wherein the nucleotide sequence of the first oligonucleotide comprisesSEQ ID NO:1.
 7. The method of claim 4, wherein the nucleotide sequenceof the first oligonucleotide consists of SEQ ID NO:1.
 8. The method ofclaim 4, wherein the nucleotide sequence of the second oligonucleotidecomprises SEQ ID NO:2.
 9. The method of claim 4, wherein the nucleotidesequence of the second oligonucleotide consists of SEQ ID NO:2.
 10. Themethod of claim 4, wherein the first and second fluorescent dyes areselected from FITC, RED640, and RED705.
 11. A method for genotyping athymidylate synthase gene of a subject, the method comprising: (a)identifying the number of tandem repeats in the promoter region of thethymidylate synthase gene by the method of claim 1, and (b) determiningthat the thymidylate synthase genotype of the subject is “homozygous2R/2R” when the number of tandem repeats is identified as two.
 12. Amethod for predicting the responsiveness of a subject toward anantitumor agent targeting thymidylate synthase, the method comprising:(a) determining the thymidylate synthase genotype of the subject by themethod of claim 11, and (b) associating the thymidylate synthasegenotype with the responsiveness of the subject towards an antitumoragent targeting thymidylate synthase.
 13. A method for determining thedose and/or the type of an antitumor agent targeting thymidylatesynthase for treating a cancer patient, the method comprising: (a)determining the thymidylate synthase genotype of the patient by themethod of claim 11, and (b) for a “homozygous 2R/2R” patient, decidingto: (i) administer an antitumor agent dose that is lower than thenormally used dose, or (ii) use an antitumor agent that has a differenttarget.
 14. The method of claim 1, wherein the nucleotide sequence ofthe second oligonucleotide comprises SEQ ID NO:2.
 15. The method ofclaim 1, wherein the nucleotide sequence of the second oligonucleotideconsists of SEQ ID NO:2.
 16. The method of claim 1, wherein the 3′ endof the first oligonucleotide is phosphorylated.
 17. A method foridentifying a number of tandem repeats in the promoter region of athymidylate synthase gene, the method comprising: (a) amplifying agenomic DNA that comprises tandem repeats in at least the promoterregion of the thymidylate synthase gene; (b) contacting, under stringenthybridization conditions, the amplified genomic DNA with either (i) afirst oligonucleotide comprising the nucleotide sequence of SEQ ID NO:1and a second oligonucleotide comprising the nucleotide sequence of SEQID NO:2, or (ii) a first oligonucleotide comprising the nucleotidesequence of the exact complement of SEQ ID NO:1 and a secondoligonucleotide comprising the nucleotide sequence of the exactcomplement of SEQ ID NO:2, wherein the hybridization conditions are suchthat hybridization between the first oligonucleotide and the amplifiedgenomic DNA can be detected if three tandem repeats are present, but notif only two tandem repeats are present; (c) detecting whetherhybridization between the first oligonucleotide and the amplifiedgenomic DNA has occurred; (d) contacting the first and secondoligonucleotides with the amplified genomic DNA of step (a) underhybridization conditions that are less stringent than in step (b); (e)detecting whether hybridization between the first oligonucleotide andthe amplified genomic DNA has occurred in (d); and (f) identifying thenumber of tandem repeats as “two” when hybridization is not detected in(c), but is detected in (e).
 18. The method of claim 17, wherein steps(c) and (e) comprise melting curve analysis.
 19. The method of claim 17,the method comprising detecting fluorescence resonance energy transferusing a first fluorescent dye at the 5′ end of the first oligonucleotideand a second fluorescent dye at the 3′ end of the secondoligonucleotide, wherein the first fluorescent dye transfersfluorescence resonance energy to the second fluorescent dye or viceversa.
 20. The method of claim 17, wherein the nucleotide sequence ofthe first oligonucleotide comprises SEQ ID NO:1.
 21. The method of claim17, wherein the nucleotide sequence of the first oligonucleotideconsists of SEQ ID NO:1.
 22. The method of claim 17, wherein thenucleotide sequence of the second oligonucleotide comprises SEQ ID NO:2.23. The method of claim 17, wherein the nucleotide sequence of thesecond oligonucleotide consists of SEQ ID NO:2.
 24. The method of claim19, wherein the nucleotide sequence of the first oligonucleotidecomprises SEQ ID NO:1.
 25. The method of claim 19, wherein thenucleotide sequence of the first oligonucleotide consists of SEQ IDNO:1.
 26. The method of claim 19, wherein the nucleotide sequence of thesecond oligonucleotide comprises SEQ ID NO:2.
 27. The method of claim19, wherein the nucleotide sequence of the second oligonucleotideconsists of SEQ ID NO:2.
 28. The method of claim 19, wherein the firstand second fluorescent dyes are selected from FITC, RED640, and RED705.29. The method of claim 17, wherein the 3′ end of the firstoligonucleotide is phosphorylated.
 30. A method for genotyping athymidylate synthase gene of a subject, the method comprising: (a)identifying the number of tandem repeats in the promoter region of thethymidylate synthase gene by the method of claim 17, and (b) determiningthat the thymidylate synthase genotype of the subject is “homozygous2R/2R” when the number of tandem repeats is identified as two.
 31. Amethod for identifying a number of tandem repeats in the promoter regionof a thymidylate synthase gene, the method comprising: (a) amplifying agenomic DNA that comprises tandem repeats in at least the promoterregion of the thymidylate synthase gene; (b) hybridizing a sample of theamplified genomic DNA with either (i) a first oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO:1 and a second oligonucleotidecomprising the nucleotide sequence of SEQ ID NO:2, or (ii) a firstoligonucleotide comprising the nucleotide sequence of the exactcomplement of SEQ ID NO:1 and a second oligonucleotide comprising thenucleotide sequence of the exact complement of SEQ ID NO:2; (c)subjecting the sample to melting curve analysis to determine the meltingtemperature (Tm); (d) comparing the Tm determined in (c) to the Tmobtained with the first oligonucleotide in each of two controls: a twotandem repeat (2R) control and a three tandem repeat (3R) control; and(e) identifying the number of tandem repeats as “two” when the Tmdetermined in (c) is more similar to the Tm obtained in the two tandemrepeat control than to the Tm obtained in the three tandem repeatcontrol.
 32. The method of claim 31, the method comprising detectingfluorescence resonance energy transfer using a first fluorescent dye atthe 5′ end of the first oligonucleotide and a second fluorescent dye atthe 3′ end of the second oligonucleotide, wherein the first fluorescentdye transfers fluorescence resonance energy to the second fluorescentdye or vice versa.
 33. The method of claim 31, wherein the nucleotidesequence of the first oligonucleotide comprises SEQ ID NO:1.
 34. Themethod of claim 31, wherein the nucleotide sequence of the firstoligonucleotide consists of SEQ ID NO:1.
 35. The method of claim 31,wherein the nucleotide sequence of the second oligonucleotide comprisesSEQ ID NO:2.
 36. The method of claim 31, wherein the nucleotide sequenceof the second oligonucleotide consists of SEQ ID NO:2.
 37. The method ofclaim 32, wherein the nucleotide sequence of the first oligonucleotidecomprises SEQ ID NO:1.
 38. The method of claim 32, wherein thenucleotide sequence of the first oligonucleotide consists of SEQ IDNO:1.
 39. The method of claim 32, wherein the nucleotide sequence of thesecond oligonucleotide comprises SEQ ID NO:2.
 40. The method of claim32, wherein the nucleotide sequence of the second oligonucleotideconsists of SEQ ID NO:2.
 41. The method of claim 32, wherein the firstand second fluorescent dyes are selected from FITC, RED640, and RED705.42. The method of claim 31, wherein the 3′ end of the firstoligonucleotide is phosphorylated.
 43. A method for genotyping athymidylate synthase gene of a subject, the method comprising: (a)identifying the number of tandem repeats in the promoter region of thethymidylate synthase gene by the method of claim 31, and (b) determiningthat the thymidylate synthase genotype of the subject is “homozygous2R/2R” when the number of tandem repeats is identified as two.
 44. Amethod for identifying a number of tandem repeats in the promoter regionof a thymidylate synthase gene, the method comprising: (a) amplifying agenomic DNA that comprises tandem repeats in at least the promoterregion of the thymidylate synthase gene; (b) hybridizing a sample of theamplified genomic DNA with either (i) a first oligonucleotide comprisingthe nucleotide sequence of SEQ ID NO:1 and a second oligonucleotidecomprising the nucleotide sequence of SEQ ID NO:2, or (ii) a firstoligonucleotide comprising the nucleotide sequence of the exactcomplement of SEQ ID NO:1 and a second oligonucleotide comprising thenucleotide sequence of the exact complement of SEQ ID NO:2; (c)subjecting the sample to melting curve analysis to determine whether thesample's melting temperature (Tm) corresponds to a two tandem repeat Tm,a three tandem repeat Tm, or a combination thereof.
 45. The method ofclaim 44, the method comprising detecting fluorescence resonance energytransfer using a first fluorescent dye at the 5′ end of the firstoligonucleotide and a second fluorescent dye at the 3′ end of the secondoligonucleotide, wherein the first fluorescent dye transfersfluorescence resonance energy to the second fluorescent dye or viceversa.
 46. The method of claim 44, wherein the nucleotide sequence ofthe first oligonucleotide comprises SEQ ID NO:1.
 47. The method of claim44, wherein the nucleotide sequence of the first oligonucleotideconsists of SEQ ID NO:1.
 48. The method of claim 44, wherein thenucleotide sequence of the second oligonucleotide comprises SEQ ID NO:2.49. The method of claim 44, wherein the nucleotide sequence of thesecond oligonucleotide consists of SEQ ID NO:2.
 50. The method of claim45, wherein the nucleotide sequence of the first oligonucleotidecomprises SEQ ID NO:1.
 51. The method of claim 45, wherein thenucleotide sequence of the first oligonucleotide consists of SEQ IDNO:1.
 52. The method of claim 45, wherein the nucleotide sequence of thesecond oligonucleotide comprises SEQ ID NO:2.
 53. The method of claim45, wherein the nucleotide sequence of the second oligonucleotideconsists of SEQ ID NO:2.
 54. The method of claim 45, wherein the firstand second fluorescent dyes are selected from FITC, RED640, and RED705.55. The method of claim 44, wherein wherein the 3′ end of the firstoligonucleotide is phosphorylated.
 56. A method for genotyping athymidylate synthase gene of a subject, the method comprising: (a)identifying the number of tandem repeats in the promoter region of thethymidylate synthase gene by the method of claim 44, and (b) determiningthat the thymidylate synthase genotype of the subject is “homozygous2R/2R” when the number of tandem repeats is identified as two.
 57. Amethod for predicting the responsiveness of a subject toward anantitumor agent targeting thymidylate synthase, the method comprising:(a) determining the thymidylate synthase genotype of the subject by themethod of claim 30, and (b) associating the thymidylate synthasegenotype with the responsiveness of the subject towards an antitumoragent targeting thymidylate synthase.
 58. A method for determining thedose and/or the type of an antitumor agent targeting thymidylatesynthase for treating a cancer patient, the method comprising: (a)determining the thymidylate synthase genotype of the patient by themethod of claim 30, and (b) for a “homozygous 2R/2R” patient, decidingto: (i) administer an antitumor agent dose that is lower than thenormally used dose, or (ii) use an antitumor agent that has a differenttarget.
 59. A method for predicting the responsiveness of a subjecttoward an antitumor agent targeting thymidylate synthase, the methodcomprising: (a) determining the thymidylate synthase genotype of thesubject by the method of claim 43, and (b) associating the thymidylatesynthase genotype with the responsiveness of the subject towards anantitumor agent targeting thymidylate synthase.
 60. A method fordetermining the dose and/or the type of an antitumor agent targetingthymidylate synthase for treating a cancer patient, the methodcomprising: (a) determining the thymidylate synthase genotype of thepatient by the method of claim 43, and (b) for a “homozygous 2R/2R”patient, deciding to: (i) administer an antitumor agent dose that islower than the normally used dose, or (ii) use an antitumor agent thathas a different target.
 61. A method for predicting the responsivenessof a subject toward an antitumor agent targeting thymidylate synthase,the method comprising: (a) determining the thymidylate synthase genotypeof the subject by the method of claim 56, and (b) associating thethymidylate synthase genotype with the responsiveness of the subjecttowards an antitumor agent targeting thymidylate synthase.
 62. A methodfor determining the dose and/or the type of an antitumor agent targetingthymidylate synthase for treating a cancer patient, the methodcomprising: (a) determining the thymidylate synthase genotype of thepatient by the method of claim 56, and (b) for a “homozygous 2R/2R”patient, deciding to: (i) administer an antitumor agent dose that islower than the normally used dose, or (ii) use an antitumor agent thathas a different target.